The identification and cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) has provided an opportunity to intercede directly in the progression of cystic fibrosis using gene therapy technologies. A number of transgenic mouse models have been developed that show varying degrees of pulmonary and gastrointestinal pathology. Highest expression of CFTR in the human has been localized to the serous cells of the submucosal glands. In addition, central bronchiectasis plays an important role in CF lung disease. The transgenic mouse CF models lack both the submucosal glands and do not develop central bronchiectasis. Porcine airway morphologic features include submucosal glands, and pigs are susceptible to many human pulmonary pathogens. The long -term goal of this project to develop the methodology for producing a porcine transgenic model for cystic fibrosis. The aim of this pilot project is to isolate lines of undifferentiated cells from porcine embryos. Such cell lines can be isolated from the inner cell mass (ICM, yielding embryonic stem cells or ES cells) and from murine primordial germ cells (PGC, yielding embryonic germ cells or EG cells). Results to date suggest that ES and EG cells are highly similar or perhaps even the same cell type. When injected into blastocysts, murine ES and EG cells are capable of differentiation into normal tissues. Porcine ES and EC cells will provide a vehicle for introducing specific changes in the genome and producing transgenic models for human disease. Despite interest in development of ES cell lines for large-animal species, documented isolation of ES cells has been limited to rodents (i.e., mouse and hamster) and possibly cattle; development of chimeric tissues after injection of putative ES cells into blastocysts has been reported only for the mouse. During the past year, two important observations were made in regard to culture of undifferentiated embryonic cells: such cell lines can be isolated from PGC as well as from the ICM, and basic fibroblast growth factor (bFGF) stimulates proliferation of undifferentiated cells in culture. Experiments are proposed in which the effect of bFGF on proliferation of cultured embryonic cells derived from porcine ICM and PCG will be tested. Cell lines that survive in culture will be tested for pluripotency by injection into blastocysts and analysis of conceptus tissues for contributions by cultured cells. Results of these experiments will be useful in establishing lines of undifferentiated porcine ES and/or EC cells for use in producing transgenic models for human disease. These results will also allow us to obtain long-term funding for this project.